A method which involves immobilizing either one of a targeted nucleic acid sample or a nucleic acid probe which is a DNA or RNA fragment comprising a nucleotide sequence complementary to a targeted nucleic acid sample, and performing hybridization between them, was developed in 1975 by E. M. Southern. This method has come to be widely used as Southern blotting (J. Mol. Biol 98, 503 (1975)). In Southern blotting, a nucleic acid sample is electrophoresed and thereafter immobilized on a nitrocellulose or nylon membrane or the like, then contacted with a nucleic acid probe having a complementary sequence.
In contrast to the above method, a method which involves immobilizing a nucleic acid probe and contacting it with a target nucleic acid to measure an amount of target nucleic acid, has also been developed and is used in gene mapping, genetic diagnosis and the like. Further, Southern et al have developed a DNA array where multiple nucleic acid probes are immobilized on a support in an array form, and have showed that DNA on a glass support binds to the complementary DNA. Further, Affymax Inc. has succeeded in developing a high densification technique of DNA array by combining Southern's technique with a DNA solid-phase synthesis technique using a photoresist method, and has commercialized this in the form of GeneChip (S. Fodor; Science 277, 393 (1997), Nature Genetics Supplement 21, 20 (1999)).
In this way, methods of detecting DNA using hybridization have been greatly advanced. However, the above mentioned methods of analysis, while being extremely effective in the detection of single stranded nucleic acid such as mRNA, when applied to the detection of double stranded nucleic acids, require a complex operation of heat-denaturing the double stranded nucleic acid present in an analyte for converting it into single strands prior to hybridization.
For example, where the subject of analysis is DNA in a genome, the subject DNA forms a double strand. For example, detection of SNP has been conducted by P. N. Gillies et al. with electric address method (Nature Biotechnology, 17, 365 (1999)) and further by R. J. Cho et al. with high-density oligo-DNA array (Nature Genetics 23, 203 (1999)). However, in either case, the determination of DNA involves a complex operation including denaturation into a single strand by heat denaturation after amplification by PCR of a target DNA portion present in a sample.
As a method for detecting double stranded DNA, use of an intercalator is known. In Japanese Patent No. 2573443, there is disclosed a method of detecting DNA using an intercalator. However, even with this method, in order to allow reaction with immobilized DNA, there is a need to firstly denature double stranded DNA into single strands.